Rett Syndrome (RS) is a neurodegenerative disorder which affects up to 1 in 15,000 liveborn females. Normal at birth, RS patients develop profound mental retardation and motor deficits in early childhood. The clinical course and pathology of RS suggest a primary defect interrupting the normal development and maintenance of specific subgroups of CNS neurons. While most cases are sporadic, familial occurrences suggest a genetic etiology with X-linked dominant inheritance and lethality or non- expression in males. Exclusion mapping using genotype analysis of familial cases excluded the putative RS gene from most of the X chromosome. Identification of the gene defect causing RS is important in that it will allow accurate diagnosis and give crucial insight toward potential treatment of RS. The primary aim of this proposal is the identification of the RS gene using four basic approaches to search for the mutation. 1) Representational Difference Analysis (RDA, a PCR-based method for isolation of unique sequences will be used to detect de novo gene rearrangements in RS patients. The likelihood of mutation detection will be maximized through the utilization of DNA from multiple unrelated families with RS probands. 2) The possibility that RS results from a triplet repeat expansion will be explored using a panel of trinucleotide repeat oligomers to identify X-linked genes containing triplet repeat sequences. Both RDA products and triplet repeat sequences will be used to screen DNA from RS patients for mutations as well as to isolate cognate cDNA or genomic clones. 3) X-linked neuronal genes will be screened for mutations by Southern and SSC Analysis. 4) Candidate X- linked genes will be examined for evidence of escape of inactivation manifest by hypomethylation and expression from the inactive S. Potential RS genes identified will be scrutinized for mutations in RS patients by DNA sequencing and examined for expression within the developing nervous system. Understanding the normal function of the gene product in development of the mammalian CNS will elucidate the molecular mechanisms underlying the pathogenesis of the RS phenotype.